Proteolytic processing of proenkephalin (PE) is required to produce active enkephalin opioid peptides that regulate analgesia, behavior, and immune cell function. It is, therefore, critical to define the multi-step proteolytic pathway required to convert PE into active enkephalin peptides. Our studies of PE processing have identified the cysteine protease termed 'prohormone thiol protease' (PTP) as the major PE cleaving activity in enkephalin-containing chromaffin granules. Notably, recent progress on this continuing project utilized active site affinity labeling and peptide microsequencing by mass spectrometry to identify the responsible PTP activity as cathepsin L. Cathepsin L is colocalized in secretory vesicles with enkephalin, and cathepsin L cleaves enkephalin-containing substrates at identical cleavage sites as native PTP. Significantly, cathepsin L knockout mice show reduced levels of enkephalin in brain. These new results implicate a key role for secretory vesicle cathepsin L in enkephalin peptide production. The cleavage specificities of cathepsin L and PTP generate peptide intermediates with NH2-terminal basic residue extensions. These findings indicate that Arg/Lys aminopeptidase is then necessary to remove such basic residues. Indeed, Arg/Lys aminopeptidase activity is colocalized in chromaffin granules with PE, enkephalin, and PTP/cathepsin L. These new discoveries of cathepsin L and Arg/Lys aminopeptidase enyzmes for PE processing complement our earlier findings showing participation of the subtilisin-like PC1 and PC2 and carboxypeptidase E/H in processing PE. These findings provide the basis for the goal of this proposal that will investigate the roles of secretory vesicle cathepsin L and Arg/Lys aminopeptidase, with PC1 and PC2, in the proteolytie pathway required for processing PE into enkephalin opioid peptides. This goal will be achieved in four specific aims, which will (1) evaluate PE processing by (a) determining the relative efficiency and cleavage sites for in vitro processing of PE by secretory vesicle cathepsin L, compared to PC1 and PC2, and (b) assessing cellular PE processing during enzyme coexpression with PE in PC12 cells, (2) assess the colocalization of cathepsin L with enkephalin and PC enzymes in chromaffin cells, transfected PC12 cells, and rat brain and neuroendocrine tissues, (3) examine the effects of reduced enzyme activity on PE processing in (a) chromaffin cells and cortical neurons subjected to antisense enzyme expression, as well as direct inhibition of cathepsin L with a selective chemical inhibitor, and in (b) cathepsin L knockout and PC2 knockout mice that show reduced enkephalin levels in brain, and (4) obtain biochemical and molecular analyses of Arg/Lys aminopeptidase for enkephalin production. Results can establish functional roles for two new protease components, secretory vesicle cathepsin L and Arg/Lys aminopeptidase, in the biosynthesis of enkephalin opioid peptides. These findings will enhance our knowledge of the complexity of the endogenous opioid system.